The actin cytoskeleton plays an essential role in basic cellular processes including locomotion, membrane transport, and cytokinesis. These processes are important for normal embryonic development, immune system function and tissue repair, and also contribute to illnesses such cardiovascular disease, cancer metastasis, and microbial infection. To enable actin function, cells precisely control the nucleation of actin polymerization and the organization of the resulting filaments. The goal of our work is to understand the function and regulation of one of the cell's key actin nucleating and organizing factors, the Arp2/3 complex. This protein complex polymerizes actin filaments into Y-branched networks that participate in lamellipodia protrusion, phagocytosis, endocytosis and membrane transport. Activation of the Arp2/3 complex requires proteins called nucleation promoting factors (NPFs), of which there are several in human cells, each acting to direct the Arp2/3 complex to participate in a particular cellular process. NPFs are themselves regulated by other cytoskeletal proteins and signal transduction molecules that coordinate their activities in space and time. However, despite the central importance of the Arp2/3 complex and its NPFs, we have yet to answer key questions about their function and regulation in vitro and in cells. For example, how is the Arp2/3 complex activated by NPFs and other interacting factors, and what role do these interactions play in the cell? Apart from a well-established role in cell migration, how do Arp2/3 complex and NPFs function in other processes such as membrane trafficking? Finally, what is the complete set of NPFs in humans, and how does each adapt the function of the Arp2/3 complex to a distinct role in the cell? To answer these questions, we propose the following specific aims. (1) We will determine how the biochemical activities of the Arp2/3 complex contribute to its cellular functions by examining the effect of Arp2/3 complex mutants on the dynamics of Y- branched actin networks in vitro and in cellular lamellipodia. (2) We will examine the function and regulation of an NPF called WHAMM, which acts in membrane transport between the ER and Golgi, by examining its mechanistic role in transport, its regulation by interacting proteins, and its ability to promote membrane dynamics in a system of purified proteins. (3) We will define the cellular function and regulation of two newly discovered NPFs called JMY and WASH by examining their localization, identifying interacting proteins and determining the cellular phenotypes caused by silencing their expression. In the long term, these experiments will help elucidate how actin nucleation and organization is controlled in cells during processes such as cell locomotion, membrane transport, and cell division. Understanding these mechanisms may ultimately lead to new approaches to diagnose and treat disease.